Protocol for NEBNext ChIP-Seq Sample Prep Master Mix Set 1 (E6240)

Protocol

  1. Starting Material: 10 ng of chromatin-immunoprecipitated (ChIP) qPCR verified or control DNA, in ≤ 40 μl of water or elution buffer.

  2. End Repair of ChIP DNA
    1. In a sterile microfuge tube mix the following components:
      ChIP DNA 1–40 μl
      NEBNext End Repair Reaction Buffer 5 μl
      NEBNext End Repair Enzyme Mix 1 μl
      Sterile H2O for a final volume of 50 μl variable
      Total volume 50 μl
    2. Incubate in a thermal cycler for 30 minutes at 20°C.
    3. Purify DNA sample on one column and elute in 44 μl of sterile dH2O or elution buffer.
  3. dA-Tailing of End Repaired DNA
    1. Mix the following components in a sterile microfuge tube:
      End Repaired DNA 44 μl
      NEBNext dA-Tailing Reaction Buffer (10X) 5 μl
      Klenow Fragment (3´→ 5´ exo) 1 μl
      Total volume 50 μl
    2. Incubate at 37°C for 30 minutes.
    3. Purify DNA sample on one column and elute in 19 μl of sterile dH2O or elution buffer.
  4. Adaptor Ligation of dA-Tailed DNA
    1. Mix the following components in a sterile microfuge tube:
      End Repaired, dA-Tailed DNA 19 μl
      Quick Ligation Reaction Buffer (5X) 6 μl
      1.5 μM DNA Adaptors* 1 μl
      Quick T4 DNA Ligase 4 μl
      Total volume 30 μl
      * Adaptors are not included, use adaptors appropriate to specific application. If necessary adjust the adaptor concentration to obtain a final adaptor to DNA molar ratio of 10:1.
    2. Incubate at 20°C for 15 minutes.
    3. Purify DNA sample on a single column and elute in 10 μl of sterile dH2O or elution buffer.
  5. Size Selection of Adaptor Ligated DNA
    1. Isolate library fragments in the 175–225 base pair range. Isolation can be performed using a number of methods including E-gel® size select gels or standard 2% agarose gels. NEB's 100bp ladder (NEB #N3231) can be used to determine the size of the fragments.
    2. If necessary, purify the DNA on a single column and elute in 36 μl of sterile water or elution buffer.
  6. PCR Enrichment of Adaptor Ligated DNA
    1. Mix the following components in a sterile microfuge tube:
      Adaptor ligated DNA 36 μl
      Phusion® HF Buffer, 5X 10 μl
      dNTP Mix 1.5 μl
      Primer 1* (25 μM stock) 1 μl
      Primer 2* (25 μM stock) 1 μl
      Phusion® High-Fidelity DNA Polymerase 0.5 μl
      Total volume 50 μl
      * Primers are not included, use primers appropriate to specific application.
    2.  PCR cycling conditions for short amplicons
      Cycle step Temp Time Cycles
      Initial denaturation 98°C 30 sec 1
      Denaturation
      Annealing
      Extension
      98°C
      65°C
      72°C
      10 sec
      30 sec
      30 sec
      18
      Final extension 72°C
      4 °C
      5 min
      hold
      1
      Purify sample on a single column and elute in 15 μl of sterile water or elution buffer.